Mouse IP-10 Gene Delivered by Folate-modified Chitosan Nanoparticles and Dendritic/tumor Cells Fusion Vaccine Effectively Inhibit the Growth of Hepatocellular Carcinoma in Mice

Abstract

Dendritic cells (DC) and tumor cell fusion vaccine (DC/tumor cell fusion vaccine) is considered an effective approach in cancer biotherapy. However, its therapeutic effects in early clinical trials have been suboptimal partially due to the immunosuppressive tumor environment. In this study, we used nanoparticles of folate (FA)-modified chitosan, a non-viral vector capable of targeting tumor cells with high expression of FA receptors. FA-chitosan nanoparticles were used as biological carriers for the expression plasmid of the mouse interferon-induced protein-10 (mIP-10) gene, a potent chemoattractant for cytotoxic T cells. The combination of FA-chitosan/mIP-10 and DC/tumor cell fusion vaccine against hepatocellular carcinoma (HCC) effectively inhibited the growth of implanted HCC tumors and prolonged the survival of mice. The combination therapy significantly reduced myeloid-derived suppressor cells (MDSC) in mouse spleen, local tumor, and bone marrow while increasing tumor-specific IFN-γ responses. Furthermore, the combination therapy significantly inhibited tumor cell proliferation while promoting their apoptosis. Taken together, our data illustrate that the mIP-10 enhances the anti-tumor effect of DC/tumor cell fusion vaccine by alleviating the immunosuppressive tumor environment.

Keywords: DC/tumor fusion cell vaccines; chitosan.; folic acid; interferon-induced protein-10.

Figure 1

Schematic of in vivo therapy using FA-chitosan/mIP-10 nanoparticles and DC/tumor fusion vaccine: FA-chitosan/mIP-10 nanoparticles are prepared by complex coacervation process and DC fusion vaccine (DC/tumor fusion cells, FC) is generated; then nanoparticles of FA-chitosan/mIP-10 and FC fusion vaccine are delivered into tumors. FA-chitosan/mIP-10 nanoparticles targeted folate receptors express and release the mIP-10 protein. Finally, mIP-10 attracts tumor-specific CTLs to kill tumor cells at the tumor site.

Figure 2

Characterization of chitosan nanoparticles. (A) Infrared spectroscopy results of nanoparticles. (B) Size distribution of nanoparticles. (C) Gel retardation assay. Lane 1: Marker; lane 2: mIP-10 plasmid; lane 3: chitosan; lane 4: chitosan/m IP-10; lane 5: FA-chitosan/mIP-10; 6: chitosanase destroyed FA-chitosan/mIP-10.

Figure 3

Identification of DC2.4/Hepa1-6 fusion cells: DC2.4 and Hepa1-6 cells were labeled with CSFE (green) and PKH26 (red), respectively, followed by fusion with PEG1450. Subsequently, the cells were stained with DAPI (blue) and observed under a fluorescent microscope. Data are representative images from three separate experiments. (A) PKH26 staining Hepa1-6 cells; (B) CFSE staining DC2.4 cells; (C) DAPI staining nuclei; (D) fused DC2.4/Hepa1-6 cells. The white arrow indicates fused cells. Original magnification, ×400.

Figure 4

FCs express higher levels of CD80, CD86 and MHCII: The levels of CD80, CD86 and MHCII expression were determined by flow cytometry and the level of IL-12 in the supernatants of cultured cells was determined by ELISA. Data are presented as histograms and expressed as the mean ± SD of individual groups of cells from three separate experiments. (A-B) Flow cytometry analysis. (C) Quantitative analysis. (D) The levels of IL-12. *** P < 0.001.

Figure 5

Treatment with FA-chitosan/mIP-10 and FC vaccine effectively inhibits the growth of implanted HCC and prolongs the survival of tumor-bearing mice. C57BL/6 mice were inoculated with Hepa1-6 cells and five days later, the mice were randomized and treated with PBS, FA-chitosan/mIP-10 and/or FC vaccine weekly for five weeks. (A) Tumor volumes. (B) Survival of tumor-bearing mice. Data are expressed as mean ± SD of the tumor volumes of each group or the percent survival of each group of mice (n = 8 mice per group). ** P < 0.01 vs. the control.

Figure 6

Treatment with FA-chitosan/mIP-10 and FC vaccine decreases the proliferation of tumor cells and increases their apoptosis in mice: At 25 days post inoculation, the mice were sacrificed and their tumor tissue sections were subjected to immunohistochemistry analysis with anti-Ki67 staining and TUNEL assays. Data are presented as representative images or expressed as the mean ± SD of each group of tumors. Immunohistochemistry analysis of Ki67⁺ proliferative tumor cells: (A) PBS; (B) FA-chitosan/mIP-10; (C) FC; (D) FA-chitosan/mIP-10 + FC; (E) Quantitative analysis. TUNEL assay of apoptotic tumor cells: (F) PBS; (G) FA-chitosan/mIP-10; (H) FC; (I) FA-chitosan/mIP-10 + FC; (J) Quantitative analysis. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 7

Treatment with FA-chitosan/mIP-10 and FC vaccine decreases the frequency of CD11b⁺LY6G⁺ granulocytic tumor infiltrates in mice: Tumor infiltrates were isolated from different groups of tumors and stained with PE-anti-LY6G and FITC-anti-CD11b. The frequency of CD11b⁺LY6G⁺ granulocytes in the tumor infiltrates was determined by flow cytometry. Data are presented as representative flow cytometry charts or expressed as the mean ± SD of each group (n = 5) of cells from three separate experiments. * P < 0.05; *** P < 0.001.

Figure 8

Treatment with FA-chitosan/mIP-10 and FC vaccine increases the frequency of tumor infiltrating CXCR3⁺CD8⁺ T cell in mice. The frequency of CXCR3⁺CD8⁺ T cells were determined by flow cytometry. Data are presented as representative images or expressed as mean ± SD of each group (n = 5) of cells from three separate experiments. (A) Flow cytometry analysis. (B) Quantitative analysis. ** P < 0.01; *** P < 0.001.

Figure 9

ELISPOT analysis of the numbers of tumor-specific IFN-γ-secreting T cells in mice: The numbers of tumor-specific IFN-γ-secreting T cells were determined by ELISPOT. Data are presented as representative images or expressed as the mean ± SD of the numbers of spot forming clones from each group of mice (n = 5) from three separate experiments. (A) PBS; (B) FA-chitosan/mIP-10; (C) FC; (D) FA-chitosan/mIP-10 + FC; (E) Quantitative analysis. ** P < 0.01; *** P < 0.001.